In the medical field, the generally preferred and most effective method of treatment is to target delivery of a pharmaceutical drug or agent to the diseased site without compromising the well being of the patient. In this regard, the importance of liposomes as drug delivery vehicles has now been recognized. Recently, liposomes have been widely used as drug carriers because they are able to function both as a controlled release system and as a delivery system for transporting encapsulated compounds to cells. More importantly, the innate ability of liposomes to reduce the toxicity of entrapped bioactive agents while maintaining efficacy is the reason why the use of liposomes has become popular. Some areas in which liposomes display therapeutic promise are as carriers for anticancer agents, antifungal agents, antibacterials, antivirals, and antiparasitics. More recently, the application of liposomes has been seen in the areas of gene therapy.
Within the cells, the majority of the liposomes are internalized through an endocytotic pathway and then subsequently enzymatically degraded. These delivery agents, when endocytosed, eventually end up in the acidic interior of endosomes having a pH between 5 and 6, and are ultimately converted into lysosomes. The acidic barrier defeats the use of liposomes as an intracellular delivery system.
Therefore, a target delivery system using pH-sensitive drug carriers is needed, wherein a pH-sensitive fusogenic system allows the payload of the delivery agent to escape from the endosomal compartment before the therapeutic agents are hydrolytically degraded. Liposomes that are dependent upon the pH of the environment are referred to as pH-sensitive liposomes.
Most pH-sensitive liposomal systems have utilized the strong tendency of unsaturated phosphatidylethanolamine (PE), as the phospholipid component, to form a non-bilayer structure and promote fusion (see, Ellens, et al., Biochemistry, 23:1532–1538 (1984)). Unsaturated PE does not form liposomes by itself, but liposomes can be prepared from unsaturated PE by incorporation of various pH-sensitive amphiphiles having a carboxyl group to stabilize the liposomal system. The stabilizing ability of these amphiphiles is designed to decrease under acidic conditions and/or cause destabilization and fusion of the liposomes.
However, this type of pH-sensitive liposome has several disadvantages. The stability of the liposomes is poor compared to phosphatidylcholine-based liposomes. For example, it has been reported that pH-sensitive liposomes composed of unsaturated PE and oleic acid (OA) rapidly aggregate and become leaky in the presence of plasma (see, Connor, et al., Biochim. Biophys. Acta., 884:474–481 (1986)). An attempt was made to increase the stability of PE/OA liposomes in serum by adding cholesterol. However, this attempt proved to be unsuccessful and as a consequence the pH sensitivity of the liposome was reduced.
More recently, polyanionic polymers having titratable functional groups attached, such as poly(2-ethylacrylic acid) (PEAA) and succinylated poly(glycidol) (see, Kono, et al., Biochim. Biophys. Acta., 1193:1–9 (1993)), have been reported to mediate proton-induced release of liposomal contents when the pH was reduced below the physiological pH. Moreover, the use of a phospholipid vesicle and the amphiphilic polyelectrolyte, poly(2-ethylacrylic acid) have previously been described in the art in U.S. Pat. No. 4,833,061. In addition, a pH-sensitive liposome was developed by immobilization of poly(2-ethylacrylic acid) on the surface of small unilamellar vesicles (SUVs) through the linking of polyelectrolytes bearing thiol functional groups to vesicles bearing maleimido functional groups, the SUV's being prepared by sonication of a dried egg phosphatidylcholine (EPC) film in buffered solutions (see, Maeda, et al. J. Am. Chem. Soc., 110:7455–7459 (1988)). However, the use of poly(2-ethylacrylic acid) conjugated to the surface of the SUVs resulted in an inconsistency or variation in the amount of entrapped calcein released upon acidification.
What is needed in the art is a pH-sensitive liposome whose fusogenicity and/or permeability can be readily controlled by a change in the pH of its environment. Preferably, this control should be engineered into the liposome architecture. This invention fulfills this and other needs.